The present invention refers broadly to the use of hemocyanin purified from the mollusk Concholepas concholepas, known as Loco, and its hemocyanin named CCH. The structure of this hemocyanin consists of two subunits with common and specific epitopes, which confer an extreme immunogenicity at the level of innate and adaptive immune responses, in all vertebrates in which it has been used. The complex molecular organization of said structure makes it an excellent carrier in the production of antibodies and the formulation of vaccines. It also gives non-specific immuno-stimulant properties when used as an immuno therapeutic agent in the treatment of some types of cancer. Furthermore it is a potent activator for natural killer cells known as NK cells.
Hemocyanins are proteins whose function is the transport of oxygen in a number of mollusk and arthropod species. These proteins contain copper which, when binding oxygen provides the protein its characteristic blue color. Hemocyanins from mollusks and arthropods have a number of applications in immunology, immunochemistry and biotechnology, since they are potent immunogens for inducing the synthesis of a variety of antibodies and also for T specific lymphocytes.
The following are some of these applications:    Use as an experimental antigen in the study of the vertebrate's immune response.    Use as a carrier protein in the production of monoclonal and polyclonal antibodies to different substances, that are not immunogenic (haptens) by themselves, such as synthetic peptides, toxins, medicines, hormones, chemical substances and recombinant microorganisms proteins and plants, animals and human proteins. These antibodies may be used in the production of diagnosis kits, in the detection of organic molecules and in human and animal therapy.    Use as a non-specific immunostimulant in the therapy of some types of cancer.    Use as a diagnosis reagent in some diseases produced by parasites like Schistosomiasis.
In this context, the best-characterized hemocyanin—almost exclusively used for the above mentioned applications—is that known as KLH, hich proceeds from the Keyhole limpet, a Californian mollusk of the species Megathura crenulata.
This expansive use of KLH has led others to seek proteins with similar immune stimulant properties. Concholepas concholepas hemocyanin has proven to be a good option in the production of monoclonal and polyclonal antibodies, for instance: against connexin 43, a gap junction protein that regulates the assembly of connexin 33 and connexin 43 in rat Sertoli cell gap junctions (Tan, I. P.; Roy, C., Sáez, J. C.; Sáez, C. G.; Paul, D. I and Risley, M. S. “Regulated assembly of connexin 33 and connexin 43 into rat Sertoli cell gap junctions”, Biology of Reproduction vol. 54, pp. 1300-131, 1996); against gizzerosin, a biogenic amine of fish meal (Becker, M. I.; Carrasco I.; Beltran, C.; Torres, M.; Jaureguiberry, B. and De loannes, A. E. “Development of monoclonal antibodies to gizzerosine, a toxic component present in fish meal”, Hybridoma vol. 17, pp. 373-381, 1998. Torres, M.; Manosalba, H.; Carrasco; De loannes, A. E and Becker, M. I. “Specific RIA for gizzerosin and simple procedure labeling method for 125I-Gizzerosin”, Journal of Agricultural and Food Chemistry vol. 47, pp. 4231-4236, 1999); against peptides (Arredondo, M.; Muñoz, P.; Mura, C. and Núñez, M. T. “HFE inhibits apical iron uptake by intestinal epithelial (Caco-2) cells”, FASEB Journal vol. 15, pp. 1276-1278, 2001. Mura, C. V.; Becker, M. I.; Orellana, A. and Wolf, D. “Immunopurification of Golgi vesicles by magnetic sorting”. Journal of Immunological Methods. vol. 260, pp. 263-71, 2002); and against marine toxin (Córdova, J. L.; Jamett, A.; Aguayo, J.; Faure, M. T., Villarroel, O and Cárdenas, L. “An in vitro assay to detect paralytic shellfish poison”, Journal Shellfish Science vol. 29, pp. 55-61, 2001).
Hemocyanin is a glycoprotein found in the hemolymph of some mollusks, which exhibits a high immunogenic capacity in vertebrates, due to its high molecular weight (between 4.5×105 and 9×107, and its phylogenetic origin widely distant from that of vertebrates.
The basic structure of hemocyanin consists in subunits organized as a decamer. In gastropods, decamers are normally associated into di-decamers, which confer them a D5 symmetry that could be considered as similar to a virus. The hemocyanin molecule contains a high number of lysine ε-amino groups, which facilitate their conjugation with other proteins and with haptens. The conjugation is carried out by means of traditional methods based on carbodiimide, glutaraldehyde, or esters of hydroxysuccinimide. One molecule of hemocyanin usually accepts up to 100 hapten molecules without losing its immunogenicity.
W. O. Weigle (“Immunochemical Properties of Hemocyanin”, Immunochemistry, vol. 1, pp. 295-302, 1964) describes the immunochemical properties of hemocyanin extracted from Megathura crenulata, however he also demonstrated that the employed preparation comprised at least two antigenic components, according to the diffusion in gel, immuno electrophoresis and electrophoresis in cellulose acetate obtained results.
J. E. Mellema and A. Klug (“Quaternary Structure of Gastropod Haemocyanin”, Nature vol. 239, pp. 146 J. E. 150, 1972) demonstrated the existence of a quaternary structure in hemocyanins extracted from three different gastropods (Kellena kelletia, Busycon canaliculatum and Helix pomatia). In all of them, the hemocyanins formed cylindrical particles, and no fundamental differences in their structure have been found.
H. B. Hercowitz, W. W. Harold and A. B. Stavitky (“Immunochemical and Immunogenic Properties of Purified Keyhole Limpet Haemocyanin”, Immunology, vol. 22, pp. 51-61, 1972) describe a highly reproducible method to obtain a relatively homogeneous preparation of hemocyanin from Megathura crenulata. In this procedure ionic exchange chromatography in DEAE-cellulose, followed by gel filtration in agarose beads is used. The product obtained was analyzed by agar immunoelectrophoresis, electrophoresis in polyacrylamide gels and by double diffusion in agar. The results show that the purified preparation contained only one main antigenic component, while the raw material contained multiple antigenic components.
J. Markl, A. Savel-Niemann. A. Wegener-Strake, A. Suling, A Schneider, W. Gebauer. J. R. Harris (“The role of two distinct subunit types in the architecture pf keyhole limpet hemocyanin [KLH]”, Naturwissenschaften, vol 78, pp. 512-514, 1991) describe the use of transmission electron microscopy with negative staining, ultra centrifugation, dissociation in adequate buffers and subsequent chromatography in polyacrylamide native gels, demonstrating that hemocyanin from Megathura crenulata contains two types of molecules: one formed by 8 functional units, named type-1, and another formed by 7 functional units, named type-2.
J. R. Harris, W. Gebauer and J. Markl (“Immunoelectron microscopy of hemocyanin from the Keyhole Limpet [Megathura crenulata]: A parallel subunit model, Journal of Structural Biology, vol 111, pp. 98-104, 1993) use monoclonal antibodies anti hemocyanin from Megathura crenulata in transmission electron microscopy with negative staining and finding that in each decamer exists an arrangement of subunits in parallel.
R. D. Swerdlow, R. F. Ebert, P. Lee, C. Bonaventura and K. I. Miller (“Keyhole limpet hemocyanin: structural and functional characterization of two different subunits and multimers”, Comparative Biochemistry and Physiology, vol. 113-B, pp. 537-548, 1996) demonstrate by immunoelectrophoresis that the two molecular forms described for hemocyanin (KLH-1 and KLH-2) do not exhibit common epitopes and differ in the immune response of experimental animals.
S. M. Söhngen, A. Stahlmann, J. R. Harris, S. A. Müller, A. Engel and J. Markl (“Mass determination, subunit organization and control of oligomerization states of keyhole limpet hemocyanin [KLH]”, European Journal of Biochemistry, vol. 248, pp. 603-614, 1997) disclose a study of the structure of KLH-1 and KLH-2 by analytic scan electronic microscopy, electrophoresis in polyacrylamide gels, immunoelectrophoresis, controlled proteolytic digestion and aminoacid sequence.
They demonstrated that these functional subunits differ in both size and the preferential aggregation form. Molecular weights of 400 KDa and 345 KDa were found for KLH-1 and KLH-2, respectively. The KLH-1 subunit has 8 different functional domains; from 45 to 65 Da. Subunit KLH-2 has 7 functional domains and lacks the C-terminal dominion named 1h, present in KLH-1. The subunits differ in their association and dissociation kinetic.
C. A. Olson, R. Chute and C. N. Rao (“Immunologic reduction of bladder cancer recurrence rate”, Journal of Urology vol. 111, pp. 173-176, 1974) disclose their observations on specific immunostimulation with Keyhole limpet hemocyanin in 29 patients (26 men and 3 women, between 30 and 93 years of age with a diagnosis of superficial transitional bladder cancer, who had not received X Ray therapy or chemotherapy). Patients were divided into two groups, according to their disease history. Group 1 was formed by 19 patients with 13 episodes of vesical tumor, and received 5 mg hemocyanin subcutaneously at the onset of the study. This group was considered as its own control, since the frequency of the tumors was known through a period of 2 years before treatment. Group 2, consisted of 19 patients with recent diagnosis (1 year) who were treated solely by transuretal resection. On the other hand, 9 patients were immunized with hemocyanin and 10 were not immunized (control group). Through a 2-year follow up, a significant reduction in the tumor recurrence frequency was found in the two groups treated with hemocyanin.
C. D. Jurincic, U. Enggelmann, J. Gasch and K. F. Klippel (“Immunotherapy in bladder cancer and Keyhole limpet hemocyanin. A randomized study” Journal of Urology, vol. 139, pp. 723-726, 1988) describe the results of two studies aimed at evaluating the immunotherapeutic effect of Keyhole limpet hemocyanin on patients with diagnosis of superficial bladder cancer. The first study, initiated in 1982, involved 44 patients who exhibited recurrent superficial bladder cancer. Previously to the therapy, the patients were immunized intracutaneously with 1 mg of hemocyanin, by means of vesical instillation with hemocyanin, and after one month they received 10 mg of hemocyanin by vesical instillation. The control group received monthly 29 mg of Mitomycin C. Twenty-one patients from the hemocyanin treated group (95.2%) exhibited partial and total prevention of the tumor, and 3 patients (14.2%) showed recurrence of the tumor, as compared with 9 patients (38.1%) of the control group. The second study started in 1984 with 81 patients who received the same treatment as in the previous study. In this case there was no control group. It was found that 17 patients (20%) showed recurrence of the tumor and 79 (86.4%) had partial and complete prevention. In the groups treated with Keyhole-limpet hemocyanin, no local or systemic adverse effects were found.
J. Flamm, A Bucher, W. HoltI and W. Albrecht (“Recurrent superficial transitional carcinoma of the bladder: Adjuvant chemotherapy versus immunotherapy. A prospective randomized trial”, Journal of Urology, vol. 144, pp. 260-263, 1990) describe the results of a comparative study on the prevention and treatment of the standard therapy of transitional bladder cancer with etoglucids versus immunotherapy with Keyhole limpet hemocyanin, in a universe of 84 patients exhibiting high risk of tumor recurrence. Prior to the onset of the instillations, all patients were submitted to a transuretal removal of the tumor; therefore, they were considered to be free of tumor when starting the treatment. The group of patients treated with etoglucid received 0.565 gm of the same weekly for six weeks and then monthly for one year. The group of patients treated with hemocyanin was immunized with 1 mg of the same intracutaneously and subsequently received vesical instillations of 30 mg during six weeks, then monthly over a year. The recurrence percentage was 60.9% in the group of patients treated with etoglucids versus 55.3% in patients treated with hemocyanin. The difference between both treatments was significant. It was concluded that the immunotherapy of this kind of recurrent tumors with hemocyanin is comparable in efficacy to that of the standard treatment.
D. L Lamm, J. I. Devane, D. R. Riggs and R. F. Ebert (“Immunotherapy of murine bladder cancer with Keyhole limpet hemocyanin (KLH)”, Journal of Urology, vol. 149, pp. 648-652, 1993) disclose the results of immunotherapy with Keyhole limpet in an experimental model of bladder cancer in mice C3H/HeN implanted with MBT2 cells, demonstrating that hemocyanin is an immunomodulator with a significant anti-tumoral activity in this animal model.
M. M. Wishahi, I. M. H. Ismail, H. Ruebben and T. Otto (“Keyhole limpet hemocyanin immunotherapy in the bilharzial bladder: A new treatment modality? Phase 2 trial; superficial bladder cancer”, Journal of Urology. Vol 153, pp. 926-928) describe the results of the treatment with Keyhole limpet hemocyanin in 13 patients who exhibited transitional bladder tumors associated with urinary schistosomiasis. The authors found that hemocyanin immunotherapy reduced the rate of tumor recurrence to 15.4% as compared with 76.9% before the therapy.
Based on the results of the technique in all the works above mentioned, hemocyanin extracted from the mollusk named Keyhole limpet (Megathura crenulata) has been traditionally used. This hemocyanin is known as KLH (Keyhole Limpet Haemocyanin).
The over exploitation of the Keyhole limpet has resulted in a scarcity of KLH in the international market. The promissory results of immunostimulation and bladder cancer immunotherapy have led to search for other molecules with similar properties. It is important to find alternative substances to replace or supplement KLH, where such new substances must present the adequate characteristics in relation to the immune response. In this context, hemocyanin from Concholepas concholepas is a good option to supplement the use of KLH.
In September 1998, Biosonda S. A. filed a Chilean patent application (Exp.No 2181-98) in the name of Alfredo Emilio De loannes lli, entitled “Purified Hemocyanin from Concholepas concholepas, procedure for its purification, formulation as immunogen and its use as an immunostimulant medicine”. Said invention consists in a hemocyanin purified for its use in vertebrates as an immunogenic agent. This hemocyanin is extracted from mollusks other than Megathura crenulata and is obtained in the form of a blue, transparent solution. In the preferred embodiment of the present invention, the mollusk used is Concholepas concholepas (Loco). Moreover a procedure is described for the purification of these hemocyanins consisting in the following operations: a. performing diagonal slits in the foot of the live mollusk, leaving the hemolymph to drain (bleed) for 30 to 60 minutes. b. submitting the hemolymph thus obtained to a saline fractioning and carrying out an ultra filtration, in order to obtain the transparent blue solution, which is rich in hemocyanin. That invention also consists in a formulation comprising hemocyanin, and to the use thereof in the immunization of vertebrates in order to prepare an immunostimulant medicine.
Oliva, H. Moltedo, B., De loannes, P. Faunes F., De loannes, A., and Becker, M. I. (“Monoclonal antibodies in molluskan hemocyanin from Concholepas concholepas demonstrate common and specific epitopes among subunits”, Hybridoma and Hybridomics vol. 21. pp. 365-374. 2002) using monoclonal antibodies specific to CCH, characterized by ELISA using different forms of CCH (dissociated in CCH-A and CCH-B subunits, by means of Western blot, enzymatic digestion, chemical deglycosylation and thermal denaturation), demonstrated that the CCH subunit displays common epitopes as well as specific epitopes for CCH-A and CCH-B subunits.